Howard Hughes Medical Institute and Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Cell. 2010 Nov 12;143(4):579-91. doi: 10.1016/j.cell.2010.10.028.
Misfolded, luminal endoplasmic reticulum (ER) proteins are retrotranslocated into the cytosol and degraded by the ubiquitin/proteasome system. This ERAD-L pathway requires a protein complex consisting of the ubiquitin ligase Hrd1p, which spans the ER membrane multiple times, and the membrane proteins Hrd3p, Usa1p, and Der1p. Here, we show that Hrd1p is the central membrane component in ERAD-L; its overexpression bypasses the need for the other components of the Hrd1p complex. Hrd1p function requires its oligomerization, which in wild-type cells is facilitated by Usa1p. Site-specific photocrosslinking indicates that, at early stages of retrotranslocation, Hrd1p interacts with a substrate segment close to the degradation signal. This interaction follows the delivery of substrate through other ERAD components, requires the presence of transmembrane segments of Hrd1p, and depends on both the ubiquitin ligase activity of Hrd1p and the function of the Cdc48p ATPase complex. Our results suggest a model for how Hrd1p promotes polypeptide movement through the ER membrane.
错误折叠的内质网腔（ER）蛋白被逆向转运到细胞质中，并被泛素/蛋白酶体系统降解。这个 ERAD-L 途径需要一个由多个内质网膜多次跨越的泛素连接酶 Hrd1p 组成的蛋白质复合物，以及膜蛋白 Hrd3p、Usa1p 和 Der1p。在这里，我们表明 Hrd1p 是 ERAD-L 中的中心膜成分；它的过表达绕过了 Hrd1p 复合物其他成分的需求。Hrd1p 的功能需要其寡聚化，而在野生型细胞中，这是由 Usa1p 促进的。位点特异性光交联表明，在逆向转运的早期阶段，Hrd1p 与接近降解信号的底物片段相互作用。这种相互作用发生在通过其他 ERAD 成分传递底物之后，需要 Hrd1p 的跨膜片段的存在，并且依赖于 Hrd1p 的泛素连接酶活性和 Cdc48p ATP 酶复合物的功能。我们的结果提出了一个模型，说明 Hrd1p 如何促进多肽通过内质网膜的运动。
